We conducted this study to evaluate the interactions of COX-2 and p53 expressions and their relations with CIMP and MSI in colorectal cancer. We demonstrated that there were inverse correlations of each of the COX-2 and p53 overexpressions with CIMP-high, and that COX-2 and p53 overexpressions were synergistically inversely associated with both CIMP-high and MSI-H. Our data imply that CIMP-low/CIMP-0 tumors tend to activate the COX-2 pathway in part through mutations of p53, a COX-2 regulator, whereas CIMP-high tumors tend not to mutate the p53
gene or to overexpress COX-2. Our data also suggest that, compared to the use of COX-2 or p53 analysis alone, the use of combined immunohistochemical analysis for both COX-2 and p53 may better serve for the molecular classification of colorectal cancer. The apparent synergistic effect of COX-2 and p53 might also reflect the limitations of immunohistochemical assays in which false positives and false negatives can occur, and the combined analysis for COX-2 and p53 might select tumors that are caused by COX-2 activation, which can, in turn, be caused by mutations and functional loss of p53. It has been shown that the correlation of TP53
gene mutations and p53 positivity by immunohistochemistry is less than perfect [44
]. Thus, the combination of COX-2 and p53 immunohistochemistry might be useful to decrease false positives and false negatives, or to select a more homogenous group of colorectal cancer cases.
We used quantitative real-time PCR (MethyLight) to distinguish high levels from low levels of DNA methylation. Assays to measure DNA methylation may be potentially very useful in clinical practice because many tumor-suppressor genes have been shown to be methylated and functionally silenced in a variety of human neoplasias [1
] DNA methylation may be a useful marker for predicting prognosis, for monitoring the efficacy of adjuvant therapy in cancer patients [45
], and for performing risk assessment in the surveillance of high-risk or low-risk individuals [46,47
]. Previous studies on CIMP in colorectal cancer [3,6–8,12
] have primarily used methylation-specific PCR (MSP). MSP is a qualitative assay and cannot reliably distinguish high levels from very low levels of methylation. Using quantitative MethyLight assays, we have shown that low levels of promoter methylation do not typically silence gene expression, suggesting that low levels of DNA methylation have no or little biologic significance [37
]. Studies using MSP may overestimate the frequency of methylation positivity in a given methylation marker, hence the frequency of CIMP. In our study, the use of quantitative DNA methylation assays and relatively unbiased samples of colorectal cancer from two large prospective cohorts has enabled us to precisely estimate the frequency of colorectal cancer cases with a specific molecular feature (i.e., CIMP, MSI-H, or Cox-2+
) at a population level.
The mechanisms of COX-2 regulation are still largely unknown. COX-2 expression has previously been shown to be repressed by wild-type p53 [30
], although p53 is unlikely the only regulator of COX-2. Thus, tumors with wild-type p53 may have a lower frequency of COX-2 overexpression. Indeed, our data support this hypothesis. The frequency of COX-2-
tumors was higher among p53-
tumors than among p53+
tumors, and COX-2 and p53 statuses were positively correlated with each other. For therapeutic approaches targeting the COX-2 pathway, it may be important to consider the effects of wild-type or mutant p53
, potentially modulating signaling through the COX-2 pathway. Further study is necessary to investigate the mechanisms of COX-2 regulation by p53.
Our data may have significant clinical implications because of the emerging importance of both COX-2 and DNA methylation as promising chemotherapeutic/chemopreventive targets. Previous studies demonstrated that MSI-H tumors, whether in sporadic or familial setting, are inversely associated with COX-2 overexpression [26,27
]. In addition, our data suggest a synergistic effect of CIMP and MSI-H on lowering the frequency of COX-2 and p53 overexpression in colorectal cancer. Elucidating the molecular mechanisms of COX-2 overexpression and its action in COX-2-overexpressed tumors and the alternative mechanisms that may bypass COX-2 overfunction in COX-2-
tumors is important for the purpose of developing molecularly targeted treatments against colorectal cancer.
The prognostic significance of CIMP stratified by COX-2 or p53 status has not been studied. Hawkins et al. [6
] showed that patients with MSS/CIMP tumors experienced worse survival compared to patients with either MSS/non-CIMP tumors or MSI-H tumors. However, their data may have been confounded by the fact that, in their study, CIMP tumors were associated with advance stage at initial presentation [6
]. In contrast, Van Rijnsoever et al. [5
] demonstrated that CIMP positivity conferred improved survival among patients who received 5-fluorouracil-based adjuvant chemotherapy for stage III colorectal cancer cases. We have previously examined promoter methylation in 25 of 34 advanced colorectal cancer cases in phase I/II clinical trials of combination chemotherapy with gefitinib (an epidermal growth factor receptor tyrosine kinase inhibitor) [48
], but we found no CIMP-high tumors (unpublished data). We found that combined positivity for both p21 and p53, but not COX-2 status, was a significant predictor of tumor resistance to combination chemotherapy with gefitinib [48
]. Further studies are necessary to determine whether CIMP stratified by COX-2 or p53 status is significantly associated with improved or worse patient outcomes. Our prospective cohort studies, the Nurses' Health Study (n
= 120,000; followed since 1976) [38
] and the Health Professional Follow-up Study (n
= 51,000; followed since 1986) [39
], are currently ongoing. Thus, relational data on patient survival and CIMP will be available in the future.
In conclusion, COX-2 and p53 overexpression exhibits synergistic inverse correlations with CIMP-high and MSI-H. Our data suggest that a combined analysis of COX-2 and p53 may be more useful for the molecular classification of colorectal cancer than either COX-2 or p53 analysis alone.